The introduction of foreign genes coding recombinant proteins into animal cells such as Chinese hamster ovary (CHO) cells can produce protein drugs for clinical treatment.
A novel erythropoiesis stimulating protein (NESP), which is an erythropoiesis-stimulating factor, is also called Darbepoetin alfa, and is a protein drug obtained by adding two N-linked sugar chains to naturally occurring erythropoietin through genetic modification (Egrie and Browne, Br. J. Cancer, 84 Suppl. 1:3-10 (2001)). NESP promotes red blood cell production by stimulating hematopoietic stem cells and facilitating their differentiation into erythrocytes. Since the serum half-life of NESP is three times longer than that of the existing recombinant erythropoietin, equivalent therapeutic effect can be expected with fewer administrations when treating anemia of patients with chronic kidney disease.
Trastuzumab as an anti-malignant tumor agent is a humanized monoclonal antibody prepared using recombinant DNA technology, and selectively acts on human epidermal growth factor receptor 2 (HER2) on cell surfaces. HER2 overexpression is confirmed in 25˜30% of primary breast cancers and trastuzumab suppresses the proliferation of HER2-overexpressed human tumor cells.
Expression levels of foreign genes may be varied depending on their insertion sites in the animal cell chromosomes since foreign gene expression is influenced by surrounding regulator elements or the chromatin structure (Zahn-Zabal et al., J. Biotechnol, 87, 29-42 (2001)).
When chromatin factors that prevent surrounding chromatins from influencing foreign gene expression are used, the suppression of gene expression depending on the position (position effect) can be overcome and the possibility of isolation of animal cell clones that highly express recombinant proteins can be increased, thereby decreasing the preparation time of cell lines for producing drugs. Attempts to produce stable cell lines by using chromatin factors that can overcome the suppression of gene expression depending on the position have been made. The factors therefor are boundary element (BE), scaffold or matrix attachment region (SAR/MAR), locus control region (LCR), and the like.
SAR also called MAR is 300˜3000-bp DNA element. It has been known that SAR allows the chromatin to attach to proteins of the nuclear matrix and controls gene expression (Makrides (Ed.), Gene Transfer and Expression in Mammalian Cells. Elsevier. Chapter 10 (2003)). In addition, SAR can improve expression of foreign genes in transfected cell lines (Poljak et al., Nucleic Acids Res, 22, 4386-4394 (1994); Kalos and Fournier, Mol. Cell. Biol, 15, 198-207 (2005)).
Zahn-Zabal et al. had produced stable cells by introducing a chicken lysozyme 5′-MAR into a luciferase expression vector and transfecting CHO cells with the vector (Zahn-Zabal et al., J. Biotechnol, 87, 29-42 (2001)). The stable cells transfected with a vector containing lysozyme 5′-MAR exhibited a higher luciferase expression level as compared with stable cells transfected with a vector not containing MAR. In addition, the vector containing two copies of MAR exhibited a higher luciferase expression level than the vector containing one copy of lysozyme 5′-MAR. This result shows that the introduction of MAR into the animal cell expression vector can increase the target protein expression level.
Korean Patent Application No. 2000-0043996 discloses that the animal cell expression vector includes human β-globin MAR so that gene expression suppression occurring when foreign genes are introduced into animal cells can be overcome. This patent describes that, besides the human β-globin MAR, human interferon-β MAR (GenBank Accession #M83137) and CSP-B 3′-SAR (GenBank Accession #M62716) are used, and these three SARs/MARs can improve the expression level of β-galactosidase, which is a target protein. Further, this patent discloses that the β-globin MAR had excellent effects as compared with the interferon-β MAR or the CSP-B 3′-SAR.
Korean Patent Application No. 2001-0079227 discloses that the introduction of human interferon-β MAR into the animal cell expression vector can improve foreign gene expression and lead to efficient expression of recombinant proteins. In addition, Korean Patent Application No. 2007-0108451 discloses that the animal cell expression vector including two copies of human β-globin MAR can further improve the target protein expression level as compared with the vector including one copy of human β-globin MAR. This patent describes comparison results between one copy and two copies of MAR factors, but not comparison results between two copies and three copies of MAR factors.
Hanson and Ley found CSP-B 5′-SAR and CSP-B 3′-SAR from approximately 70 kb of the human chromosome 14q11.2 hematopoietic serine protease gene cluster and obtained their nucleotide sequences, and verified that these SARs bind to scaffolds derived from the cellular nucleus through in vitro experiment (Hanson and Ley, Blood, 79, 610-618 (1992)).
The CSP-B 5′-SAR and CSP-B 3′-SAR are registered as GenBank Accession #M62717 and GenBank Accession #M62716, respectively. They are 2429-bp long and 1233-bp long, respectively, and thus the CSP-B 5′-SAR is two folds longer than the 3′-SAR. Human chromosome 14 DNA sequence is registered as GenBank Accession #AL136018. In this sequence, the length between a transcription initiation codon for a gene coding the CSP-B protein and the 5′-SAR located upstream thereof is 1195 bp, and the length between a transcription termination codon of the gene for coding the CSP-B protein and the 3′-SAR located downstream thereof is 4543 bp.
Throughout the entire specification, many papers and patent documents are referenced and their citations are represented. The disclosures of cited papers and patent documents are entirely incorporated by reference into the present specification, and the level of the technical field within which the present invention falls and details of the present invention are explained more clearly.